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Click Chemistry for the Identification of G-Quadruplex Structures Discovery of a DNAЦRNA G-Quadruplex.

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Angewandte
Chemie
DOI: 10.1002/ange.200806306
Biological Structures
Click Chemistry for the Identification of G-Quadruplex Structures:
Discovery of a DNA–RNA G-Quadruplex**
Yan Xu,* Yuta Suzuki, and Makoto Komiyama*
Four-stranded DNA structures formed by guanine-rich
sequences are known as G-quadruplexes. They have received
much interest over the last decade owing to their remarkable
structural features and biological importance.[1] For example,
G-quadruplex DNA composed of telomeric sequences plays
an important role in telomere maintenance and has become a
potential tumor-selective target for chemotherapy.[2] Various
studies based on platinum cross-linking, fluorescence resonant energy transfer (FRET), 125I radioprobing, covalent
ligation, sedimentation, NMR spectroscopy, and X-ray crystallography, among other techniques, have been carried out to
investigate G-quadruplex structures.[3] For example, by using
a photochemical method, we detected the diagonal loops in
an antiparallel G-quadruplex.[4] Although these approaches
gave some structural information, the development of a more
effective method based on a chemical reaction for probing Gquadruplex structures would be desirable. Ideally, a chemical
reaction to distinguish a complex G-quadruplex structure
should be mild, highly selective, almost quantitative, and
readily initiated and quenched. Click chemistry, a classification for powerful and selective reactions, may fulfill all the
necessary criteria.[5]
Herein, we describe the application of the coppercatalyzed azide–alkyne cycloaddition (CuAAC), the most
extensively studied “click reaction”,[6] to explore G-quadruplex solution structures. Click chemistry has previously been
used to functionalize viruses, proteins, and oligonucleotides,
as well as to immobilize DNA on electrode surfaces and
chips.[7] A phosphodiester linkage within the loop position of
a G-quadruplex has also been used to synthesize circular
oligonucleotides for exploring G-quadruplex structure.[8] We
now report that a copper-catalyzed cycloaddition occurs in
different G-quadruplex structures between azido and alkyne
groups located at the 5’ and 3’ ends of the G-quadruplex. The
use of this simple “click” method enables the detection of the
[*] Dr. Y. Xu, Y. Suzuki, Prof. M. Komiyama
Research Center for Advanced Science and Technology
The University of Tokyo
Komaba, Meguro-ku, Tokyo 153-8904 (Japan)
Fax: (+ 81) 3-5254-5201
E-mail: xuyan@mkomi.rcast.u-tokyo.ac.jp
komiyama@mkomi.rcast.u-tokyo.ac.jp
Homepage: http://www.mkomi.rcast.u-tokyo.ac.jp/index.html
[**] This research was partially supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, Sports, Culture
and Technology of Japan. Support by the Global COE Program for
Chemistry Innovation through the Cooperation of Science and
Engineering is also acknowledged.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200806306.
Angew. Chem. 2009, 121, 3331 –3334
G-quadruplex structure. Importantly, by using this approach
we discovered a DNA–RNA hybrid-type G-quadruplex
structure formed from human telomeric DNA and RNA
sequences. G-quadruplexes often exist in equilibrium with
one another, and it is often impossible to determine the kind
of species present in solution. Whereas FRET and other
methods, such as NMR spectroscopy, give an averaged signal
(depending on the time scale of interconversion in the case of
NMR spectroscopy), the click method enables a snapshot to
be taken of the existing species in solution. The species
trapped by the click reaction can be separated and analyzed.
This approach will greatly facilitate G-quandruplex analysis
in solution under various conditions.
To determine whether a click reaction could occur on a Gquadruplex, we first employed a parallel dimeric telomere
RNA G-quadruplex used in our recent studies (Figure 1 a).[9]
Figure 1. a) The parallel dimeric telomere RNA G-quadruplex formed
by 12-mer telomere RNA. b) The antiparallel dimeric G-quadruplex
formed by the 12-mer Oxytricha nova sequence d(G4T4G4).
We prepared the 12-mer 2’-OMe oligoribonucleotides
(ORNs) ORN-1, with a 5’-alkyne, and ORN-2, with a 5’azido group (Figure 2 a; see the Experimental Section for the
sequences). Analysis by denaturing gel electrophoresis of the
click reaction of ORN-1 and ORN-2 for 1 h at room
temperature under the conditions of G-quadruplex formation
(200 mm KCl) showed that no reaction occurred without the
copper catalyst (Figure 2 b, lane 1). In the presence of the
copper catalyst, a new band of lower mobility appeared
(lanes 2–4). The appearance of this band is attributed to the
reaction between the 5’-alkyne of ORN-1 and the 5’-azido
group of ORN-2 to form linear ORN-3, which was purified by
reversed-phase (RP) HPLC and characterized by MALDITOF MS (Figure 2 c). To further demonstrate that the click
reaction was promoted by G-quadruplex formation, we
prepared the 12-mer ORN-4 (rKU12) without the telomeric
sequence. No reaction occurred (Figure 2 b, lane 5) under the
conditions used for the reactions analyzed in lanes 2–4. This
result indicates clearly that the formation of the G-quadruplex promotes the click reaction by bringing the 5’-alkyne
2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Zuschriften
Figure 2. a) Click reaction on a human telomeric RNA G-quadruplex.
b) Analysis of the click reaction of ORN-1 and ORN-2 by denaturing
gel electrophoresis. Lane 1: without the Cu catalyst. Lanes 2–4: with
the Cu catalyst. The molar ratio of ORN to Cu was 1:25 (lane 2), 1:50
(lane 3), and 1:100 (lane 4). ORN-1 and ORN-2 (lower band) are
gradually consumed to give ORN-3 (upper band). Lane 5: with the Cu
catalyst and with ORN-4 (without the telomere sequence) in place of
ORN-2. c) MALDI-TOF MS spectrum of ORN-3 (m/z 8733.5
[M H+Na]+).
and 5’-azido reaction partners into close proximity to one
another.
We next employed an antiparallel G-quadruplex formed
by an Oxytricha nova sequence to investigate the possibility of
click reactions on different types of G-quadruplex structure
(Figure 1 b).[10] The 5’-alkyne- and 3’-azido-labeled 12-mer
oligodeoxyribonucleotide (ODN) ODN-5 was used (Figure 3 a). Without the copper catalyst, no reaction occurred
(Figure 3 b, lane 1). Interestingly, in the presence of the
copper catalyst, two new bands appeared (lane 2). The
upper band, with a position between those of the 20-mer
and 30-mer oligonucleotide markers, was thought to be a
click-reaction product, 24C: a 24-mer circular oligonucleotide. The bottom band in lane 2 was a self-cyclization product,
Figure 3. a) Click reaction between a 5’-alkyne and a 3’-azido group on
the G-quadruplex of the Oxytricha nova sequence. b) Denaturing gel
electrophoresis of the click-reaction products. Lane M: 30-, 20-, and
10-mer oligonucleotide markers; lane 1: without the Cu catalyst;
lane 2: with the Cu catalyst; lane 3: ODN-6 as a reference. L: linear
DNA, C: circular DNA.
3332
www.angewandte.de
12C, which was identified by comparison with the gel mobility
of the reference oligonucleotide ODN-6 in lane 3 (Figure 3 b).
These results suggested that click reactions can occur on DNA
and RNA G-quadruplex scaffolds. The click ligation reaction
on the G-quadruplex is very mild and straightforward. It was
carried out in water at room temperature for a reaction time
of only 1 h. The reaction is highly selective and can be
initiated simply by adding the copper catalyst and quenched
by gel filtration.
Having established this efficient click reaction on RNA
and DNA G-quadruplexes, we applied this method to the
analysis of G-quadruplex structures. A three-repeat and a
single-repeat human telomeric sequence are known to form a
(3 + 1) dimeric DNA G-quadruplex in the presence of
NaCl.[11] Although it has been speculated that these two
oligonucleotides could form the same structure in a K+
solution, which would represent physiological conditions
more closely, critical evidence is required (Figure 4 a).[12] We
Figure 4. Schematic representation of two possible G-quadruplex
structures: a) a (3 + 1) dimeric DNA G-quadruplex formed by 16-mer
and 6-mer human telomeric sequences; b) a DNA–RNA hybrid Gquadruplex formed by 12-mer human telomere DNA and 12-mer
human telomere RNA.
used the method developed in the current study to determine
whether the (3 + 1) dimeric DNA G-quadruplex can be
formed in the presence of KCl. For this purpose, we
synthesized the 5’-alkyne- and 3’-azido-labeled 16-mer oligonucleotide ODN-7, and the 5’-azido- and 3’-alkyne-labeled 6mer oligonucleotide ODN-8 (Figure 5 a; see Schemes S2 and
S3 in the Supporting Information). A click reaction between
ODN-7 and ODN-8 was performed in the presence of KCl
and analyzed by polyacrylamide gel electrophoresis (Figure 5 b). When the copper catalyst was added, a new product
(lane 2, 22C), likely to be the circular oligonucleotide derived
from the two linear precursors, was generated from the click
reaction. This result indicates that the formation of a (3 + 1)
dimeric DNA G-quadruplex between ODN-7 and ODN-8
promotes the click reaction to form the circular oligonucleotide.
To obtain further evidence of the formation of the (3 + 1)
dimeric G-quadruplex in KCl solution, we prepared a 22-mer
circular ODN containing a photocleavable linker (Figure 6 a)
from the 6-mer ODN (containing the photocleavable linker)
and the 16-mer ODN-7. Upon irradiation with UV light, the
22-mer circular ODN 22C should dissociate to give the 22mer linear ODN 22L (Figure 6 a), whereas a linear ODN
would produce two linear ODNs. The photocleavable circular
oligonucleotide was irradiated with UV light for 15 min
2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. 2009, 121, 3331 –3334
Angewandte
Chemie
Figure 5. Confirmation of the formation of a (3 + 1) dimeric DNA Gquadruplex by a click reaction in a solution containing K+ ions. a) Click
reactions between the 5’-alkyne and the 5’-azido group and between
the 3’-azido group and the 3’-alkyne lead to a 22-mer circular
oligonucleotide. b) Analysis of the click reaction between ODN-7 and
ODN-8 by denaturing gel electrophoresis. Lane 1: without the Cu
catalyst; lane 2: with the Cu catalyst; lane 3: ODN-9 as a reference.
Figure 6. Further evidence for the formation of a (3 + 1) dimeric DNA
G-quadruplex in a solution containing K+ ions. a) The photocleavable
linker in a circular ODN, 22C, is cleaved by UV irradiation to give a
linear ODN, 22L. b) Analysis by denaturing gel electrophoresis of the
dissociation of the circular oligonucleotide through cleavage by UV
irradiation. Lane 1: without the Cu catalyst and without UV irradiation;
lane 2: with the Cu catalyst without UV irradiation; lane 3: with the Cu
catalyst and UV irradiation. The new band, 22L, in lane 3 is the linear
oligonucleotide produced by the cleavage of 22C with UV light.
(Figure 6 b). As expected, only one new band (lane 3, 22L)
appeared, with a similar mobility to that of the circular ODN
22C. Without UV irradiation, no new band appeared (Figure 6 b, lane 2). We could therefore conclude that the product
of the click reaction was the circular ODN 22C.
The products of the click reaction were characterized
further by RP HPLC and MALDI-TOF MS (see Figure S1 in
the Supporting Information). The main two peaks with
different retention times (tR(ODN-9) = 18.1 min; tR(ODN10) = 19.5 min) from those of the starting materials (tR(ODN7) = 17.6 min; tR(ODN-8) = 20.8 min) were characterized by
MALDI-TOF MS (see Figure S1 in the Supporting Information), which revealed that the peaks corresponded to the
Angew. Chem. 2009, 121, 3331 –3334
circular product ODN-10 (m/z 8050.9 [M H+Na]+) and the
self-cyclization product ODN-9.
Recently, two studies demonstrated that telomeres are
transcribed into telomeric repeat-containing RNA in mammalian cells.[13, 14] Telomeric RNAs containing mainly
UUAGGG repeats of heterogeneous length were detected
in different human and rodent cell lines. This discovery of
telomere RNA raises the crucial question of how telomeric
RNA is specifically associated with telomeric DNA in terms
of chromosome-end regulation and protection. Telomere
RNAs were found to be localized with the telomere
DNA,[13, 14] which suggests a possible association between
telomere RNA and telomere DNA. We suspect that telomere
RNA may bind to telomere DNA through the formation of an
intermolecular DNA–RNA G-quadruplex (Figure 4 b),
although no experimental data have yet been obtained that
demonstrate the existence of the DNA–RNA hybrid Gquadruplex structure directly. It is technically difficult to
study the DNA–RNA hybrid G-quadruplex structure by
traditional methods, such as NMR spectroscopy and X-ray
crystallography, since the DNA G-quadruplex, the RNA Gquadruplex, and the DNA–RNA hybrid G-quadruplex may
coexist as a mixture in solution. In fact, we have demonstrated
that human telomere RNA can form a parallel G-quadruplex
structure in the presence of sodium[9] and had difficulty in
studying the DNA–RNA hybrid structure by NMR spectroscopy (unpublished data).
We believed that click chemistry might be a useful method
for the detection of a DNA–RNA hybrid G-quadruplex. We
designed a click reaction in which only the DNA–RNA hybrid
G-quadruplex could undergo an azide–alkyne cycloaddition,
even in the presence of the corresponding DNA–DNA or
RNA–RNA dimeric G-quadruplex (Figure 7 a). A 5’-azido
ODN, ODN-12, and a 5’-alkyne-labeled ORN, ORN-1, were
prepared as substrates for the click reaction. With a copper
catalyst, a new band appeared with a mobility shift between
that of the 20-mer oligonucleotide marker and that of the 30mer marker (Figure 7 b, lane 1). This band is thought to be the
product of a click reaction between the 5’-alkyne-labeled
RNA and the 5’-azido DNA in a DNA–RNA hybrid Gquadruplex. Moreover, the 5’-alkyne-labeled 12-mer ORN-4
without the telomere sequence and 5’-azido ODN-12 were
subjected to the same reaction conditions; no reaction
occurred (lane 2), which suggests that DNA–RNA G-quadruplex formation is required for the click reaction. This result
indicates strongly that human telomere 12-mer DNA and 12mer RNA can form a hybrid-type G-quadruplex.
To the best of our knowledge, it has not been reported
previously that a G-quadruplex can act as a scaffold for a click
reaction. Reactions of a 5’ alkyne with a 5’ azide, of a 3’ alkyne
with a 3’ azide, and of a 5’ alkyne with a 3’ azide can occur in
different types of G-quadruplex structures. We used this
method to probe the structure of G-quadruplexes and showed
that the (3 + 1) dimeric DNA G-quadruplex can form in the
presence of KCl. Most importantly, by using this approach, we
discovered a DNA–RNA hybrid-type G-quadruplex structure
formed by human telomeric DNA and RNA sequences. The
advantage of the click reaction is that the detection of the
reaction products identifies the G-quadruplex structure
2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.de
3333
Zuschriften
Table 1: Oligonucleotides used in this study.[a]
Name
Sequence
ORN-1
ORN-2
ORN-3
ORN-4
ODN-5
ODN-6
ODN-7
ODN-8
ODN-9
ODN-10
ODN-11
ODN-12
5’-KUAGGGUUAGGGU-3’
5’-ZUAGGGUUAGGGU-3’
(3’-UGGGAUUGGGAU-5’)X(5’-UAGGGUUAGGGU-3’)
5’-KUUUUUUUUUUUU-3’
5’-KGGGGTTTTGGGGZ-3’
oligonucleotide derived from ODN-5 by self-cyclization
5’-KGGGTTAGGGTTAGGGTZ-3’
5’-ZTAGGGTK-3’
oligonucleotide derived from ODN-7 by self-cyclization
circular oligonucleotide formed from ODN-7 and ODN-8
5’-ZTAGGGTPK-3’
5’-ZTAGGGTTAGGGT-3’
[a] The ORNs are composed of 2’-OMe RNA. K = alkyne, Z = azide, P =
photocleavable linker, X = 1,2,3-triazole.
Figure 7. Detection of a DNA–RNA hybrid G-quadruplex by a click
reaction. a) Schematic depiction of the detection of the DNA–RNA Gquadruplex. The use of 5’-azido-labeled DNA and 5’-alkyne-labeled
RNA may result in a mixture of three types of G-quadruplex. Only the
DNA–RNA G-quadruplex brings the alkyne and the azido group into
close proximity to give the product of an azide–alkyne cycloaddition.
b) Analysis of the click reaction of the 5’-azido ODN and the 5’-alkyne
ORN by denaturing gel electrophoresis. Lane M: 30-, 20-, and 10-mer
oligonucleotide markers; lane 1: with the Cu catalyst; lane 2: with the
Cu catalyst and with ORN-4 in place of ORN-1.
[4]
[5]
[6]
[7]
directly in a complex solution, whereas traditional methods,
such as NMR spectroscopy and X-ray crystallography, may
not be suitable.
Experimental Section
The oligonucleotides used in this study (Table 1) were prepared
according to Schemes S1–S3 in the Supporting Information.
Received: December 24, 2008
Published online: March 30, 2009
.
Keywords: click chemistry · cycloaddition · DNA structures ·
G-quadruplexes · RNA structures
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